Ethanol is a commonly encountered toxic substance. Methods for qualitative and quantitative determination of ethanol in body fluids, particularly human body fluids, are used in medicine and in law enforcement. In medicine, the level of ethanol in the blood is significant in diagnosing liver malfunction and alcoholism, as well as for understanding the reason for an emergency room patient being comatose. In law enforcement, such assays are used to determine whether or not an automobile operator is driving under the influence of alcohol.
Ethanol testing can be accomplished using both enzymatic and nonenzymatic assays. The nonenzymatic assays have a number of disadvantages and are being widely replaced by enzymatic assays which are more accurate, highly specific, more sensitive and require less expensive procedures. Enzymatic assays are generally based on the use of alcohol dehydrogenase to catalyze the reaction of ethanol to acetaldehyde. This reaction can be used alone, or in combination with other reactions to produce a spectrophotometric signal which can be related to the amount of ethanol in the tested specimen.
One enzymatic assay is based on the direct measurement of the reduced coenzyme (NADH), such as that described in U.S. Pat. No. 3,926,736 (Bucolo). This assay is carried out entirely in solution.
Another enzymatic assay is described in EP-A-0 464 942 (published Jan. 1, 1992) which uses nicotinamide adenine dinucleotide (AND.sup.+) as a coenzyme with alcohol dehydrogenase to produce the reduced form of the coenzyme. The coenzyme, in turn, reacts with a tetrazolium salt to produce a detectable dye. The described assay is carried out in a multilayer analytical element containing tris(hydroxymethyl)aminomethane buffer and both crosslinked and uncrosslinked gelatin layers.
One problem that has been encountered in developing a dry analytical element for the assay of ethanol is the strong interference by fluoride ion present in human serum. Fluoride ion is commonly used as a preservative in serum, and interferes in assays possibly by altering the equilibrium between ethanol and acetaldehyde, and causes the assay results to be biased positively compared to the true value of ethanol in the specimen.
There is a great need for a sensitive and accurate assay for ethanol which can be carried out using an analytical element, which assay is not affected by fluoride ion.